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L0301P38 - Gene Expression
Differential Gene Expression *all cells in a eukaryote have the same genes but they are expressed differentially *difference in protein production is due to differential transcription **selective transcription of specific genes **transcriptional control/regulation Regulation of Transcription Protein Binding *many different proteins involved in initiating transcription **RNA polymerase **general transcription factors ***help binding of RNA polymerase **specific transcription factors ***help modulate transcription by binding to enhancer or repressor regions Promoter Sequence *determines site of transcription initiation *generally upstream from start of the DNA sequence to be transcribed *binding site of RNA Polymerase II and transcription apparatus Enhancer Sequence *activator proteins bind to them stimulating transcription *have a variable location - can be up to 50,000b up/downstream of promoter within an intron *can contain multiple clustered binding sites *some enhancer sequences are used only in specific cell types   Repressor/Silencer Sequence *repressors are regulatory proteins (transcription factors) that act to prevent the production of the protein *may function by: **competing with activators for binding sites on DNA **masking activator activity **having a direct negative interaction on the transcription complex assembled at the promoter Overall control of the rate and level of transcription is achieved by combinations of proteins. DNA-Protein Interaction *proteins that directly bind DNA have a DNA- binding region whose protein surface is partially “complementary” to the surface features of the DNA recognition site *DNA recognition sites are generally short stretches (>20bp) but of high specificity **recognise the sequence, and/or the major or minor groove Transcription Factors Motifs in Transcription Factors *are short, recurring patterns in DNA that often indicate sequence-specific binding sites for proteins (DNA-protein interaction) Types *Helix-turn-helix - involved in development *Leucine zipper - regulate cell division genes *Zinc finger - steroid hormone receptors *Helix-loop-helix - regulate immune system genes Regulation of Transcription Factors *As they are also proteins, TF activity can be controlled by: **protein synthesis **ligand binding **protein phosphorylation **addition of a second subunit **unmasking (exposure of active site) **stimulation of nuclear entry **release from membrane RNA Involvement in Gene Regulation MicroRNAs *a.k.a. miRNAs *short, 19-25 nucleotide, non-coding RNAs *negatively regulate gene expression through sequence-specific base-pairing with mRNA targets *are expressed in a tissue-specific fashion Other small RNAs *small interfering RNA (siRNA) **regulate activity of transposons, viral infection and a few protein coding genes *Piwi interacting RNA (piRNA) **essential for development of germ cells (sperm in mammals)   Chromatin Involvement in Gene Expression Chromatin Structure *dynamic, dense packaging of DNA to fit within a cell *packaging of DNA hinders the accessibility of proteins that are required for gene expression Permanent Silencing of Transcription *occurs in DNA of higher-order forms of chromatin *inactive chromatin is assumed to contain proteins that render the DNA unusually inaccessible *permanent in telomeres Reversible Silencing of Transcription *reversible in other regions of DNA to allow gene activation to occur Involves: 1. histone modifications *histones have N-terminal tails rich in basic amino acids which extend from the surface of the nucleosome *histone can be altered¹ by acetylation and removed by other proteins so transcription can occur ¹there are numerous other ways histones can be modified including methylation, phosphorylation, ubiquitination, glycosylation 2. ATP-dependent chromatin remodelling *chromatin remodelling complexes bring about changes in structure of chromatin *use energy of ATP hydrolysis to bind DNA and move histone octomers of nucleosomes *in general, the affinity for DNA- and chromatin- associated proteins is controlled by modification and positioning of histones H2A/H2B/H3/H4 Histone Acetylation *acetylation of all 4 core histones (H2A, H2B, H3, H4) occurs on conserved lysines *reversible, controlled by enzymes: **acetylate = histone acetyltransferases **deacetylate = histone deacetylases *hyperacetylation = activation of transcription *hypoacetylation = inactivation/silencing  Limiting Gene Activation Chromatin Loops *insulator binding proteins isolates the loop of DNA that is active allowing selective gene expression Barrier Proteins *tether regions of chromosomes to fixed sites to prevent the spread of gene activation DNA Methylation Important functions in mammals: *control of gene expression *cellular differentiation and development (programs of gene expression) *preservation of chromosomal integrity *X-chromosome inactivation in females *Implicated in brain function and development of the immune system *alterations in genomic patterns contribute to human cancers Methylation Patterns *occurs at the 5’ position of cytosine at CG islands *does not affect base pairing *genomic pattern is inherited during DNA replication *methylation is stable but reversible and maintained by methyltransferases *epigenetic - methylation is “stamped” on the DNA without changing its sequence *generally both alleles are methylated in the same way Transcriptional Control *methylated Cs found in clusters within or near the promoter of 60% of genes *tight binding of proteins (MeCBPs) to clustered methylated C’s correlates to transcriptional repression *histone deacetylation binds to methyl C binding proteins causing more repression Genomic Imprinting *epigenetic phenomenon by which certain genes are expressed in a parent-of-origin-specific manner *in mammals, the maternal and paternal genomes are both required for normal embryonic and postnatal development *for imprinted genes, there is a difference in expression depending on whether it was inherited form the mother or father **it is believed that the gene from one parent is “silenced” by methylation **if the allele inherited from the father is imprinted, it is thereby silenced, and only the allele from the mother is expressed Implications Disease may result if: *the active parental copy is affected by mutation (in the simplest case there are now two inactive gene copies) *mutation occurs in genes controlling imprinting (too much silencing) *genes that encode activators or repressors are imprinted leading to altered expression of their target genes   Changes in the DNA ‘methylome’ *Hypomethylation may led to: **chromosome instability **activation of endogenous parasitic sequences **loss of imprinting *Local hypermethylation may lead to: **inactivation of key cell pathways in DNA repair, cell cycle control (tumour suppressor genes) *either case can lead to cancer Epigenetic Programming During Development Gametogenesis *de novo methylation gives rise to substantially methylated genomes in sperm and eggs *sex specific differences are evident in the level and in the pattern of methylation Early Embryo *In the early embryo soon after fertilisation there is genome wide de-methylation *From pre-gastrulation onwards the extent of methylation varies: **somatic cell lineages ***heavy methylation **trophoblast-derived lineages ***giving rise to placenta, yolk sac ***less methylated **primordial germ cells ***largely unmethylated until cycle repeats Imprinting and Reproductive Cloning *genes in the donor nucleus do not pass through a germ line state before they find themselves back at the beginning of development – not imprinted appropriately *this imprinting “failure” is probably a major reason why animal cloning is so inefficient *imprinting “failures” are not also known to have implications for fitness of IVF embryos **higher cancer rates, developmental problems   X-Chromosome Inactivation *one of the X chromosomes in each cell of a female is inactivated early in development *methylation of cytosine on DNA is involved *the inactive X has one gene, Xist, that is only lightly methylated and transcriptionally active *RNA (interference) transcribed from Xist is a non-coding RNA and remains in the nucleus *it binds to the X chromosome that transcribes it and triggers inactivation  